Canine CD20 Compositions

ABSTRACT

The invention provides canine CD20 nucleotide and protein sequences. These compositions are useful in the diagnosis and treatment of, e.g., CD20+ B-cell lymphoma, immune-mediated hemolytic anemia, immune-mediated thrombocytopenia, and systemic lupus erythematosus (SLE) in canines and felines.

PRIORITY

This application is a divisional application of U.S. application Ser.No. 11/138,949, filed May 26, 2005, which claims the benefit of U.S.Application 60/575,172 filed May 28, 2004, which are incorporated hereinby reference in their entirety.

BACKGROUND OF THE INVENTION

CD20 is a transmembrane protein that is expressed on more than 95% ofB-lymphocytes. Expression at the cell surface occurs from the pre-Bstage of development until differentiation to a plasma cell. The proteinhas several functions; it serves as a calcium channel, it is involved inintracellular signal transduction, and it can modulate cell growth anddifferentiation. In human medicine, anti-CD20 monoclonal antibodytherapeutics (e.g.: Rituxan (Rituximab) by Genentech and IDECPharmaceuticals) have been successfully utilized to treat relapsed orrefractory low-grade or follicular, CD20+, B-cell non-Hodgkin's lymphoma(NHL). Rituxan has also been used in treating immune-mediated hemolyticanemia, immune-mediated thrombocytopenia, and systemic lupuserythematosus (SLE). Rituxan, however, does not bind canine B cells.See, Impellizeri et al., Vet. Cancer Society. 2003. Proceed. 23^(rd)Ann. Conf., p. 2.

SUMMARY OF THE INVENTION

The invention provides canine CD20 nucleotide and protein sequences.These compositions are useful in the diagnosis and treatment of, e.g.,CD20+ B-cell lymphoma, immune-mediated hemolytic anemia, immune-mediatedthrombocytopenia, and systemic lupus erythematosus (SLE) in canines andfelines.

One embodiment of the invention provides an isolated canine CD20 proteincomprising SEQ ID NO:6.

Another embodiment of the invention provides a polypeptide comprisingSEQ ID NO:6, wherein the polypeptide has one or more amino acidsubstitutions at about one to about 45 positions selected from aminoacid positions 8, 9, 11, 12, 14, 15, 16, 17, 19, 21, 24, 25, 26, 27, 29,30, 31, 32, 34, 35, 37, 43, 47, 51, 65, 73, 74, 75, 76, 77, 82, 83, 84,94, 102, 105, 106, 108, 109, 112, 116, 118, 121, 131, 133, 134, 139,141, 142, 143, 147, 150, 151, 153, 154, 155, 156, 157, 158, 159, 162,163, 165, 166, 168, 170, 171, 172, 178, 180, 184, 187, 189, 192, 193,194, 195, 196, 198, 199, 200, 201, 202, 205, 208, 218, 219, 221, 222,223, 225, 226, 227, 228, 232, 234, 235, 240, 241, 242, 243, 244, 247,248, 249, 251, 252, 253, 254, 259, 262, 270, 275, 277, 280, 282, 285,288, 290, 292, 295, 296, and 297, wherein the polypeptide is not SEQ IDNO:7, SEQ ID NO:8, or SEQ ID NO:9 and wherein the polypeptide isisolated, purified and about 297 to about 300 amino acids long. Thepolypeptide can further comprise one or more amino acid sequences thatare not canine amino acid sequences. The one or more amino acidsubstitutions can be conservative amino acid substitutions. The aminoacid substitutions can be selected from the following substitutions:Amino Acid Amino Acid Position Substitution 8 V 9 N 11 P 12 F 14 A 15 E16 A 17 T 19 G 21 L, I 24 Q, N 25 S 26 G, A 27 P 29 P, V 30 L, N 31 F, L32 R 34 T 35 S 37 L 43 S 47 K 51 A, P 64 M 65 T 73 P 74 A, M 75 G, E 76I 77 F 82 L, V, M 83 S 84 V 94 Y 102 V 105 A 106 E 108 T 109 S 112 C 116A 118 V 121 S 131 M 133 L 134 S 139 L 141 M 142 K, A 143 L 147 F 150 R151 S, R 153 E 154 F 155 L 156 R, Q 157 T, S 158 H, S 159 T, K 162 I 163N 165 Y 166 D, T 168 E, Q 170 S 171 K 172 S 178 P 180 T 184 Y, N, D 187Q 189 L 192 S 193 I 194 L 195 S 196 A 198 L 199 V 200 S 201 A 202 L 205E 208 I 218 R 219 T, M 221 T 222 R 223 S 225 A 226 N 227 DELETE 228 I232 S 234 G 235 N 240 T, L 241 V 242 K 243 I, M 244 K 247 V, I, A 248 I249 G 251 S 252 G 253 T, V 254 S 259 N 262 E 270 I 275 T, A 277 T, M 280P 282 A 285 D 288 P 290 L 292 V 295 E 296 I 297 S, AThe polypeptide comprises one or more of the following amino acidadditions: an A after amino acid 104; an E after amino acid 169, and anE after amino acid 274.

Even another embodiment of the invention provides an isolatedpolypeptide comprising SEQ ID NO:10. The polypeptide can be present in afusion protein.

Still another embodiment of the invention provides a polypeptidecomprising SEQ ID NO:10, wherein the polypeptide has about one to about12 amino acid substitutions at positions 3, 5, 6, 7, 14, 15, 17, 18, 19,20, 21, 22, 23, 26, 27, 29, 30, 32, 35, and 45, wherein the polypeptideis not SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14, and wherein thepolypeptide is isolated, purified and is about 53 amino acids long. Theamino acid substitutions can be conservative amino acid substitutions.The amino acid substitutions can be selected from the followingsubstitutions: Amino Acid Position Substitution 3 L 5 M 6 K, A 7 L 11 F14 R 15 S, R 17 E 18 F 19 L 20 R, Q 21 T, S 22 H, S 23 T, K 26 I 27 N 29Y 30 D, T 32 E, Q 34 S 35 K 36 S 42 P 44 T 45 K 48 N, Y, D 51 Q 53 LThe polypeptide can comprise one or more amino acid sequences that arenot canine amino acid sequences. The polypeptide can comprise an aminoacid addition of E after amino acid number 33. The polypeptide cancomprise SEQ ID NO:1.

Yet another embodiment of the invention provides an antibody or antigenbinding portion thereof that specifically binds SEQ ID NO:6 or SEQ IDNO:10. The isolated antibody or antigen binding portion thereof can be amonoclonal antibody, a polyclonal antibody, or single chain antibody.The antibody can be produced by myeloma cell line ATCC PTA-6661 or ATCCPTA-6662. The antigen binding portion thereof can be a Fab fragment, aF(ab′)₂ fragment, or a Fv fragment.

Another embodiment of the invention provides an isolated polynucleotidethat encodes a canine CD20 protein. The polynucleotide can comprise SEQID NO:5. The isolated polynucleotide can comprise about 12 or morecontiguous nucleic acids of SEQ ID NO:5.

Even another embodiment of the invention provides an isolatedpolynucleotide that encodes the extracellular domain of a canine CD20protein.

Yet another embodiment of the invention provides a vector comprising apolynucleotide of the invention and a recombinant host cell thatcomprises a vector of the invention.

Still another embodiment of the invention provides a method of producinga recombinant cell that expresses a canine CD20 protein, or fragmentthereof, comprising transfecting a cell with the vector of theinvention. A canine CD20 polypeptide or a fragment thereof can beproduces by expressing the polypeptide in the recombinant host cell ifthe invention.

Another embodiment of the invention provides a method of detecting acanine or feline CD20-positive B-lymphocyte. The method comprisescontacting one or more antibodies that specifically bind to apolypeptide consisting of SEQ ID NO:6 or SEQ ID NO:10 with a test sampleunder conditions that allow B-lymphocyte/antibody complexes to form anddetecting B-lymphocyte/antibody complexes. The detection ofB-lymphocyte/antibody complexes is an indication that a canine or felineCD20-positive B-lymphocyte is present in the sample and the absence ofB-lymphocyte/antibody complexes is an indication that a canine or felineCD20-positive B-lymphocyte is not present in the sample. The one or moreantibodies can be monoclonal antibodies, polyclonal antibodies, orantibody fragments. The sample can be lymph node aspirate, serum, orwhole blood.

Yet another embodiment of the invention provides a method of treating acanine or feline for CD20-positive B-cell lymphoma, immune-mediatedhemolytic anemia, immune-mediated thrombocytopenia, or systemic lupuserythematosus (SLE) comprising administering an antibody of theinvention to the canine or feline.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a portion of the nucleotide sequence for canine CD20 fromcanine peripheral blood mononuclear cell cDNA (SEQ ID NO:1).

FIG. 2 shows the translated polypeptide (SEQ ID NO:4) of SEQ ID NO:1aligned with comparable regions of human CD 20 (SEQ ID NO:2) and mouseCD20 (SEQ ID NO:3).

FIG. 3 shows the full-length polynucleotide of canine CD20 (SEQ ID NO:5)

FIG. 4 shows the full-length polypeptide sequence (SEQ ID NO:6) forcanine CD20 The amino acid sequence was deduced from cDNA sequenceanalysis.

FIG. 5 shows the alignment of the full length canine CD20 polypeptide(SEQ ID NO:6) with that of human (SEQ ID NO:7), mouse (SEQ ID NO:8) andcat (SEQ ID NO:9). The canine sequence is most similar to the felinesequence (84% identical) and less similar to that of human (74%identical) and mouse (68% identical).

FIG. 6 is an anti-FLAG Western blot demonstrating expression of canineCD20 in COS7 cells. Lane 1 is an empty plasmid control, lane 2 is totalcell lysate of cells transiently transfected with canine CD20, lanes 3-8are total cell lysates from transfected cells immunoprecipitated witheither anti-FLAG (3), anti-CD20 monoclonals (4,5), or anti-CD20polyclonal antibodies (6-8).

FIG. 7 is an example of a COS7 cell expressing canine CD20 on thesurface of the cell as detected by anti-FLAG immunofluoresence andconfocal microscopy with three-dimensional reconstruction.

FIG. 8 shows a 53-mer polypeptide of the predominant extracellulardomain of canine CD20 (SEQ ID NO:10) that was synthesized alone and inconjunction with a murine T-cell epitope from ovalbumin (SEQ ID NO:11).

FIG. 9 depicts the serum titers obtained from two mice immunized withcanine CD20 as evaluated in the peptide ELISA.

FIG. 10 is a Coomassie stained SDS-PAGE gel showing the purified IgMmonoclonal antibodies that recognize the extracellular domain of canineCD20. Monoclonal antibody F3C7 is shown in lane 1, F7A5 is shown in lane2, and F19 is shown in lane 3.

FIG. 11 depicts the flow cytometry results for the four fluorescentlylabeled IgM monoclonal antibodies to canine CD20 when used to label alymph node aspirate from dogs with lymphoma.

FIG. 12 shows the flow cytometry results for the fluorescently labeledIgM monoclonal antibody F7A5 on a lymph node aspirate from a dog with aB-cell lymphoma. Scatter plots are also shown for an isotype control forthe CD20 antibody (IgM-fluorescein), an anti-CD79a B-cell antibody andthe corresponding IgG1 isotype control.

FIG. 13 shows the scatter plots obtained using the colloidal goldlabeled F7A5 monoclonal antibody on a lymph node aspirate from a normaldog as analyzed by a point of care hematology instrument. Panel Adepicts the unlabeled control and F7A5 labeled cells for gating on smalllymphocytes, while panel B depicts the same comparison when gating onmedium lymphocytes.

FIG. 14 depicts an example of how the colloidal gold labeled F7A5monoclonal antibody can be used on a point of care hematology instrumentto detect CD20-positive B-lymphocytes in a cat with lymphoma.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides

A polypeptide is a polymer of three or more amino acids covalentlylinked by amide bonds. A polypeptide can be post-translationallymodified. A purified polypeptide is a polypeptide preparation that issubstantially free of cellular material, other types of polypeptides,chemical precursors, chemicals used in synthesis of the polypeptide, orcombinations thereof. A polypeptide preparation that is substantiallyfree of cellular material, culture medium, chemical precursors,chemicals used in synthesis of the polypeptide has less than about 30%,20%, 10%, 5%, 1% or more of other polypeptides, culture medium, chemicalprecursors, and/or other chemicals used in synthesis. Therefore, apurified polypeptide is about 70%, 80%, 90%, 95%, 99% or more pure.

Polypeptides of the invention comprise full-length canine CD20 andfragments thereof. One embodiment of the invention provides an isolatedpolypeptide comprising SEQ ID NO:6 or SEQ ID NO:10. Another embodimentof the invention provides a polypeptide comprising SEQ ID NO:10 andhaving amino acid substitutions, for example, conservative amino acidsubstitutions, at one or more positions (for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 24 or more amino acidsubstitutions) selected from amino acid positions 3, 5, 6, 7, 14, 15,17, 18, 19, 20, 21, 22, 23, 26, 27, 29, 30, 32, 35, and 45 of SEQ IDNO:10, wherein the polypeptide is not SEQ ID NO:12 or SEQ ID NO:13, orSEQ ID NO:14 and wherein the polypeptide is isolated, purified and isabout 53 amino acids long. An amino acid addition of “E” can occur afteramino acid number 33. In one embodiment of the invention SEQ ID NO:10has one or more substituted amino acids (for example, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 24 amino acid substitutions) asshown in Table 1 and the polypeptide is not SEQ ID NO:12, SEQ ID NO:13,or SEQ ID NO:14 and the polypeptide is isolated, purified and about 53amino acids long. TABLE 1 Amino Acid Position Substitution 3 L 5 M 6 K,A 7 L 11 F 14 R 15 S, R 17 E 18 F 19 L 20 R, Q 21 T, S 22 H, S 23 T, K26 I 27 N 29 Y 30 D, T 32 E, Q 34 S 35 K 36 S 42 P 44 T 45 K 48 N, Y, D51 Q 53 L

Another embodiment of the invention provides a polypeptide comprisingSEQ ID NO:6 having amino acid substitutions, for example, conservativeamino acid substitutions, at one or more positions (e.g., about 1, 2, 3,4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more amino acidsubstitutions) selected from amino acid positions 8, 9, 44, 12, 14, 15,16, 17, 19, 21, 24, 25, 26, 27, 29, 30, 31, 32, 34, 35, 37, 43, 47, 51,65, 73, 74, 75, 76, 77, 82, 83, 84, 94, 102, 105, 106, 108, 109, 112,116, 118, 121, 131, 133, 134, 139, 141, 142, 143, 147, 150, 151, 153,154, 155, 156, 157, 158, 159, 162, 163, 165, 166, 168, 170, 171, 172,178, 180, 184, 187, 189, 192, 193, 194, 195, 196, 198, 199, 200, 201,202, 205, 208, 218, 219, 221, 222, 223, 225, 226, 227, 228, 232, 234,235, 240, 241, 242, 243, 244, 247, 248, 249, 251, 252, 253, 254, 259,262, 270, 275, 277, 280, 282, 285, 288, 290, 292, 295, 296, 297 whereinthe polypeptide is not SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9 andwherein the polypeptide is isolated, purified, and about 297 to about299 amino acids long. In one embodiment of the invention SEQ ID NO:6 hasone or more (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60 or more) substituted amino acids as shown in Table 2 and thepolypeptide is not SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9 and thepolypeptide is isolated, purified and about 297 to about 300 amino acidslong. Additionally, amino acid additions can occur as follows: an Aafter amino acid 104; an E after amino acid 169, an E after amino acid274 of SEQ ID NO:6. TABLE 2 Amino Acid Amino Acid Position Substitution8 V 9 N 11 P 12 F 14 A 15 E 16 A 17 T 19 G 21 L, I 24 Q, N 25 S 26 G, A27 P 29 P, V 30 L, N 31 F, L 32 R 34 T 35 S 37 L 43 S 47 K 51 A, P 64 M65 T 73 P 74 A, M 75 G, E 76 I 77 F 82 L, V, M 83 S 84 V 94 Y 102 V 105A 106 E 108 T 109 S 112 C 116 A 118 V 121 S 131 M 133 L 134 S 139 L 141M 142 K, A 143 L 147 F 150 R 151 S, R 153 E 154 F 155 L 156 R, Q 157 T,S 158 H, S 159 T, K 162 I 163 N 165 Y 166 D, T 168 E, Q 170 S 171 K 172S 178 P 180 T 184 Y, N, D 187 Q 189 L 192 S 193 I 194 L 195 S 196 A 198L 199 V 200 S 201 A 202 L 205 E 208 I 218 R 219 T, M 221 T 222 R 223 S225 A 226 N 227 DELETE 228 I 232 S 234 G 235 N 240 T, L 241 V 242 K 243I, M 244 K 247 V, I, A 248 I 249 G 251 S 252 G 253 T, V 254 S 259 N 262E 270 I 275 T, A 277 T, M 280 P 282 A 285 D 288 P 290 L 292 V 295 E 296I 297 S, A

The basic and novel characteristics of polypeptides of the inventionthat consist essentially of SEQ ID NO:6 or SEQ ID NO:10 are that theyconsist essentially of the sequences shown in SEQ ID NO:6 and SEQ IDNO:10 and that they specifically bind to an antibody, antibody fragment,or single-chain antibody of the invention.

One of skill in the art would expect that amino acid substations and/oradditions could be made in the amino acid sequences of SEQ ID NO:6 orSEQ ID NO:10 as described above, wherein the amino acid sequences wouldretain their functional activity. For example, a F7A5 monoclonalantibody (see Example 6), which is specific for SEQ ID NO:10specifically binds both feline and canine CD20-positive B lymphocytes(see Example 7 and 8), despite the fact that sequences of feline andcanine CD20 are not 100% homologous.

An isolated polypeptide is a molecule of amino acids that is notimmediately contiguous with one or both canine flanking amino acidsequences that the molecule is normally associated with in nature. Anisolated polypeptide is also a molecule of amino acids that forms partof a hybrid polypeptide comprising additional non-canine polypeptidesequences that can be, for example, a fusion protein.

The invention also includes functionally active variants of SEQ ID NO:6and SEQ ID NO:10. Functionally active variants of SEQ ID NO:6 or SEQ IDNO:10 can comprise one or more of the amino acid substitutions asdescribed above. In one embodiment, a functionally active variantpolypeptide includes an amino acid sequence at least about 60% identicalto a sequence shown as SEQ ID NO:6 and SEQ ID NO:10. Preferably, thepolypeptide is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99% or more identical to SEQ ID NO:6 or SEQ ID NO:10 andspecifically binds to an antibody of the invention. Functionally activevariant polypeptides and polypeptides of the invention also specificallybind to an antibody, such as a monoclonal antibody, that is raised to apolypeptide shown in SEQ ID NO:6 or SEQ ID NO:10.

Polypeptides of the invention specifically bind to an antibody of theinvention. In this context “specifically binds” means that thepolypeptide recognizes and binds to an antibody of the invention withgreater affinity than to other, non-specific molecules. For example, anantibody raised against an antigen (polypeptide) to which it binds moreefficiently than to a non-specific protein can be described asspecifically binding to the antigen. Binding specifically can be testedusing, for example, an enzyme-linked immunosorbant assay (ELISA), aradioimmunoassay (RIA), or a western blot assay using methodology wellknown in the art.

A polypeptide is a functionally active variant if it reactssubstantially the same as a polypeptide shown in SEQ ID NO:6 or SEQ IDNO:10 in an assay such as an immunohistochemical assay, an ELISA, anRIA, or a western blot assay, e.g. has 90-110% of the specific bindingactivity of the original polypeptide. In one embodiment, the assay is acompetition assay wherein the functionally active variant polypeptide iscapable of reducing binding of a polypeptide shown in SEQ ID NO:6 or SEQID NO:10 to a corresponding antibody, antibody fragment, or single-chainantibody by about 80, 95, 99, or 100%.

Functionally active variants can also comprise “polypeptide fragments”of the invention. Polypeptide fragments comprise or consist essentiallyof about 15, 20, 30, 40, 50, 100, 150, 200, or 299 amino acids of SEQ IDNO:6 or SEQ ID NO:10.

As used herein, percent identity of two amino acid sequences (or of twonucleic acid sequences) is determined using the algorithm of Karlin andAltschul (PNAS USA 87:2264-2268, 1990), modified as in Karlin andAltschul, PNAS USA 90:5873-5877, 1993). Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al. (J.Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches are performedwith the NBLAST program, score=100, wordlength=12. BLAST proteinsearches are performed with the XBLAST program, score=50, wordlength=3.To obtain gapped alignment for comparison purposes GappedBLAST isutilized as described in Altschul et al. (Nucleic Acids Res.25:3389-3402, 1997). When utilizing BLAST and GappedBLAST programs thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)are used to obtain nucleotide sequences homologous to a nucleic acidmolecule of the invention.

Identity or identical means amino acid sequence (or nucleic acidsequence) similarity and has an art recognized meaning. Sequences withidentity share identical or similar amino acids (or nucleic acids).Thus, a candidate sequence sharing 85% amino acid sequence identity witha reference sequence requires that, following alignment of the candidatesequence with the reference sequence, 85% of the amino acids in thecandidate sequence are identical to the corresponding amino acids in thereference sequence, and/or constitute conservative amino acid changes.

Functionally active variants of SEQ ID NO:6 or SEQ ID NO: 10 retainsubstantially the same functional activity of the original polypeptideor fragment. Naturally occurring functionally active variants such asallelic variants and species variants and non-naturally occurringfunctionally active variants are included in the invention and can beproduced by, for example, mutagenesis techniques or by direct synthesis.

A functionally active variant differs by about, for example, 1, 2, 3, 4,5, 10, 20, 30, 40, 50, 60 or more amino acid residues from a polypeptideshown in SEQ ID NO:6 or SEQ ID NO:10 or a fragment thereof. Where thiscomparison requires alignment the sequences are aligned for maximumhomology. The site of variation can occur anywhere in the polypeptide,as long as activity substantially similar to a polypeptide shown in SEQID NO:6 and SEQ ID NO:10 is maintained.

Guidance concerning how to make phenotypically silent amino acidsubstitutions is provided in Bowie et al., Science, 247:1306-1310(1990), which teaches that there are two main strategies for studyingthe tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, the amino acid positions which havebeen conserved between species can be identified. See e.g., FIG. 5.These conserved amino acids are likely important for protein function.In contrast, the amino acid positions in which substitutions have beentolerated by natural selection indicate positions which are not criticalfor protein function. Thus, positions tolerating amino acid substitutioncan be modified while still maintaining specific binding activity of thepolypeptide.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site-directed mutagenesis oralanine-scanning mutagenesis (the introduction of single alaninemutations at every residue in the molecule) can be used (Cunningham etal., Science, 244:1081-1085 (1989)). The resulting variant molecules canthen be tested for specific binding to antibodies of the invention.

According to Bowie et al., these two strategies have revealed thatproteins are surprisingly tolerant of amino acid substitutions. Theauthors further indicate which amino acid changes are likely to bepermissive at certain amino acid positions in the protein. For example,the most buried or interior (within the tertiary structure of theprotein) amino acid residues require nonpolar side chains, whereas fewfeatures of surface or exterior side chains are generally conserved.

Methods of introducing a mutation into amino acids of a protein is wellknown to those skilled in the art. See, e.g., Ausubel (ed.), CurrentProtocols in Molecular Biology, John Wiley and Sons, Inc. (1994); T.Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1989)).Mutations can also be introduced using commercially available kits suchas “QuikChange™ Site-Directed Mutagenesis Kit” (Stratagene). Thegeneration of a polypeptide functionally active variant to a polypeptideby replacing an amino acid that does not influence the function of apolypeptide can be accomplished by one skilled in the art.

In one embodiment of the invention, a polypeptide of the invention isderived from a canine. A polypeptide of the invention can be isolatedfrom cells or tissue sources using standard protein purificationtechniques. Polypeptides of the invention can also be synthesizedchemically or produced by recombinant DNA techniques. For example, apolypeptide of the invention can be synthesized using conventionalpeptide synthesizers. Additionally, a polynucleotide encoding apolypeptide of the invention can be introduced into an expression vectorthat can be expressed in a suitable expression system using techniqueswell known in the art. A variety of bacterial, yeast, plant, mammalian,and insect expression systems are available in the art and any suchexpression system can be used. Optionally, a polynucleotide encoding apolypeptide of the invention can be translated in a cell-freetranslation system.

A functionally active variant polypeptide can also be isolated using ahybridization technique. Briefly, DNA having a high homology to thewhole or part of a nucleic acid sequence encoding SEQ ID NO:6 or SEQ IDNO:10 is used to prepare a functionally active polypeptide. Therefore, apolypeptide of the invention also includes polypeptides that arefunctionally equivalent to a SEQ ID NO:6 or SEQ ID NO:10 polypeptide andare encoded by a nucleic acid molecule that hybridizes with a nucleicacid encoding SEQ ID NO:6 or SEQ ID NO:10 or a complement thereof. Oneof skill in the art can easily determine nucleic acid sequences thatencode polypeptides of the invention using readily available codontables. As such, these nucleic acid sequences are not presented herein.

The stringency of hybridization for a nucleic acid encoding apolypeptide that is a functionally active variant is, for example, 10%formamide, 5×SSPE, 1× Denhart's solution, and 1× salmon sperm DNA (lowstringency conditions). More preferable conditions are, 25% formamide,5×SSPE, 1× Denhart's solution, and 1× salmon sperm DNA (moderatestringency conditions), and even more preferable conditions are, 50%formamide, 5×SSPE, 1× Denhart's solution, and 1× salmon sperm DNA (highstringency conditions). However, several factors influence thestringency of hybridization other than the above-described formamideconcentration, and one skilled in the art can suitably select thesefactors to accomplish a similar stringency.

Nucleic acid molecules encoding a functionally active variantpolypeptide can also be isolated by a gene amplification method such asPCR using a portion of a nucleic acid molecule DNA encoding apolypeptide shown in SEQ ID NO:6 or SEQ ID NO: 10 as the probe.

Polypeptides of the invention can also comprise those that arise as aresult of the existence of multiple genes, alternative transcriptionevents, alternative RNA splicing events, and alternative translationaland postranslational events. A polypeptide can be expressed in systems,e.g., cultured cells, which result in substantially the samepostranslational modifications present as when the polypeptide isexpressed in a native cell, or in systems that result in the alterationor omission of postranslational modifications, e.g., glycosylation orcleavage, present when expressed in a native cell.

A conservative substitution is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. In general, the following groups of amino acids representconservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr;(2) cys, ser, tyr, tlr; (3) val, ile, leu, met, ala, phe; (4) lys, arg,his; and (5) phe, tyr, trp, his.

A polypeptide of the invention can further comprise a signal (or leader)sequence that co-translationally or post-translationally directstransfer of the protein. The polypeptide can also comprise a linker orother sequence for ease of synthesis, purification or identification ofthe polypeptide (e.g., poly-His), or to enhance binding of thepolypeptide to a solid support. For example, a polypeptide can beconjugated to an immunoglobulin Fc region or bovine serum albumin.

A polypeptide can be covalently or non-covalently linked to an aminoacid sequence to which the polypeptide is not normally associated within nature. Additionally, a polypeptide can be covalently ornon-covalently linked to compounds or molecules other than amino acids.For example, a polypeptide can be linked to an indicator reagent, anamino acid spacer, an amino acid linker, a signal sequence, a stoptransfer sequence, a transmembrane domain, a protein purificationligand, or a combination thereof. In one embodiment of the invention aprotein purification ligand can be one or more C amino acid residues at,for example, the amino terminus or carboxy terminus of a polypeptide ofthe invention. An amino acid spacer is a sequence of amino acids thatare not usually associated with a polypeptide of the invention innature. An amino acid spacer can comprise about 1, 5, 10, 20, 100, or1,000 amino acids.

If desired, a polypeptide can be a fusion protein, which can alsocontain other amino acid sequences, such as amino acid linkers, aminoacid spacers, signal sequences, TMR stop transfer sequences,transmembrane domains, as well as ligands useful in proteinpurification, such as glutathione-5-transferase, histidine tag, andstaphylococcal protein A, or combinations thereof. A fusion protein istwo or more different amino acid sequences operably linked to eachother. A fusion protein construct can be synthesized chemically usingorganic compound synthesis techniques by joining individual polypeptidefragments together in fixed sequence. A fusion protein construct canalso be expressed by a genetically modified host cell (such as E. coli)cultured in vitro, which carries an introduced expression vector bearingspecified recombinant DNA sequences encoding the amino acids residues inproper sequence. The heterologous polypeptide can be fused, for example,to the N-terminus or C-terminus of a polypeptide of the invention. Apolypeptide of the invention can also comprise homologous amino acidsequences, i.e., other CD20 or CD20-derived sequences. More than onepolypeptide of the invention can be present in a fusion protein.Fragments of polypeptides of the invention can be present in a fusionprotein of the invention. A fusion protein of the invention cancomprise, e.g., one or more of SEQ ID NO:6, SEQ ID NO:10, fragmentsthereof, or combinations thereof.

Polypeptides of the invention can be in a multimeric form. That is, apolypeptide can comprise one or more copies of SEQ ID NO:6, SEQ ID NO:10or a combination thereof. A multimeric polypeptide can be a multipleantigen peptide (MAP). See e.g., Tam, J. Immunol. Methods, 196:17-32(1996).

Polypeptides of the invention can comprise an antigen that is recognizedby an antibody reactive against canine CD20. The antigen can compriseone or more epitopes (i.e., antigenic determinants). An epitope can be alinear epitope, sequential epitope or a conformational epitope. Epitopeswithin a polypeptide of the invention can be identified by severalmethods. See, e.g., U.S. Pat. No. 4,554,101; Jameson & Wolf, CABIOS4:181-186 (1988). For example, a polypeptide of the invention can beisolated and screened. A series of short peptides, which together spanan entire polypeptide sequence, can be prepared by proteolytic cleavage.By starting with, for example, 100-mer polypeptide fragments, eachfragment can be tested for the presence of epitopes recognized in anELISA. For example, in an ELISA assay a canine CD20 polypeptide, such asa 100 mer polypeptide fragment, is attached to a solid support, such asthe wells of a plastic multi-well plate. A population of antibodies arelabeled, added to the solid support and allowed to bind to the unlabeledantigen, under conditions where non-specific absorption is blocked, andany unbound antibody and other proteins are washed away. Antibodybinding is detected by, for example, a reaction that converts acolorless substrate into a colored reaction product. Progressivelysmaller and overlapping fragments can then be tested from an identified100-mer to map the epitope of interest.

A polypeptide of the invention can be produced recombinantly. Apolynucleotide encoding a polypeptide of the invention can be introducedinto a recombinant expression vector, which can be expressed in asuitable expression host cell system using techniques well known in theart. A variety of bacterial, yeast, plant, mammalian, and insectexpression systems are available in the art and any such expressionsystem can be used. Optionally, a polynucleotide encoding a polypeptidecan be translated in a cell-free translation system. A polypeptide canalso be chemically synthesized or obtained from E. ewingii cells.

An immunogenic polypeptide of the invention can comprise an amino acidsequence shown in, e.g., SEQ ID NO:6 or SEQ ID NO:10. An immunogenicpolypeptide can elicit antibodies or other immune responses (e.g.,T-cell responses of the immune system) that recognize epitopes ofpolypeptides having SEQ ID NO:6 or SEQ ID NO:10. An immunogenicpolypeptide of the invention can also be a fragment of a polypeptidethat has an amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:10. Animmunogenic polypeptide fragment of the invention can be about 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids inlength.

Polynucleotides

Polynucleotides of the invention contain less than an entire caninegenome and can be single- or double-stranded nucleic acids. Apolynucleotide can be RNA, DNA, cDNA, genomic DNA, chemicallysynthesized RNA or DNA or combinations thereof. The polynucleotides canbe purified free of other components, such as proteins, lipids and otherpolynucleotides. For example, the polynucleotide can be 50%, 75%, 90%,95%, 96%, 97%, 98%, 99%, or 100% pure by dry weight. Purity can bemeasured by a method such as column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis. The polynucleotides of the inventionencode the polypeptides described above. In one embodiment of theinvention the polynucleotides encode polypeptides shown in, e.g., SEQ IDNO:6 or SEQ ID NO:10 or combinations thereof. Polynucleotides of theinvention can comprise other nucleotide sequences, such as sequencescoding for linkers, signal sequences, TMR stop transfer sequences,transmembrane domains, or ligands useful in protein purification such asglutathione-S-transferase, histidine tag, and staphylococcal protein A.

The polynucleotides of the invention encode the polypeptides describedabove, as well as fragments thereof. A fragment can be about 10, 12, 15,20, 50, 75, 100, 125, 250, 300, 400, 500, 600, 700, 800, 900, 1,000 ormore polynucleotides. One of skill in the art can obtain thepolynucleotide sequence of the invention using the disclosed polypeptidesequence and codon tables. Polynucleotides can contain naturallyoccurring polynucleotides or sequences that differ from those of anynaturally occurring sequences or polynucleotides. In one embodiment ofthe invention, a polynucleotide of the invention is derived from amammal, such as a dog. Polynucleotides of the invention can differ fromnaturally occurring nucleic acids, but still encode naturally occurringamino acids due to the degeneracy of the genetic code. Polynucleotidesof the invention can also comprise other heterologous nucleotidesequences, such as sequences coding for linkers, signal sequences, aminoacid spacers, heterologous signal sequences, TMR stop transfersequences, transmembrane domains, or ligands useful in proteinpurification such as glutathione-5-transferase, histidine tag, andstaphylococcal protein A. Polynucleotides of the invention can alsocomprise other homologous nucleotide sequences, i.e., other CD20 orCD20-derived sequences.

An isolated polynucleotide is a nucleic acid molecule that is notimmediately contiguous with one or both of the 5′ and 3′ flankingsequences with which it is normally contiguous when present in anaturally occurring genome. Therefore, an isolated polynucleotide canbe, for example, a polynucleotide that is incorporated into a vector,such as a plasmid or viral vector, a polynucleotide that is incorporatedinto the genome of a heterologous cell (or the genome of a homologouscell, but at a site different from that where it naturally occurs); anda polynucleotide that exists as a separate molecule such as apolynucleotide produced by PCR amplification, chemically synthesis,restriction enzyme digestion, or in vitro transcription. An isolatedpolynucleotide is also a nucleic acid molecule, such as a recombinantnucleic acid molecule that forms part of hybrid polynucleotide encodingadditional polypeptide sequences that can be used for example, in theproduction of a fusion protein.

A polynucleotide can also comprise one or more expression controlsequences such as promoters or enhancers, for example. A polynucleotideof the invention can be present in a vector, such as, for example, anexpression vector. If desired, polynucleotides can be cloned into anexpression vector comprising, for example, promoters, enhancers, orother expression control sequences that drive expression of thepolynucleotides of the invention in host cells. The polynucleotides canbe operably linked to the expression control sequences. An expressionvector can be, for example, a plasmid, such as pBR322, pUC, or ColE1, oran adenovirus vector, such as an adenovirus Type 2 vector or Type 5vector. Vectors suitable for use in the present invention include, forexample, bacterial vectors, mammalian vectors, viral vectors (such asretroviral, adenoviral, adeno-associated viral, herpes virus, simianvirus 40 (SV40), and bovine papilloma virus vectors) andbaculoviris-derived vectors for use in insect cells. Polynucleotides insuch vectors are preferably operably linked to a promoter, which isselected based on, e.g., the cell type in which expression is sought.

Host cells into which vectors, such as expression vectors, comprisingpolynucleotides of the invention can be introduced include, for example,prokaryotic cells (e.g., bacterial cells) and eukaryotic cells (e.g.,yeast cells; insect cells; and mammalian cells). Such host cells areavailable from a number of different sources that are known to thoseskilled in the art, e.g., the American Type Culture Collection (ATCC),Rockville, Md. Host cells into which the polynucleotides of theinvention have been introduced, as well as their progeny, even if notidentical to the parental cells, due to mutations, are included in theinvention.

Methods for introducing polynucleotides of the invention (e.g., vectorscomprising the polynucleotides or naked polynucleotides) into cells,either transiently or stably, are well known in the art. For example,transformation methods using standard CaCl₂, MgCl₂, or RbCl methods,protoplast fusion methods or transfection of naked or encapsulatednucleic acids using calcium phosphate precipitation, microinjection,viral infection, and electroporation.

One embodiment of the invention provides methods of producing arecombinant cell that expresses a canine CD20 protein, or fragmentthereof, comprising transfecting a cell with a vector comprising thepolynucleotide of the invention. A canine CD20 protein, or fragmentthereof, can then be produced by expressing the polypeptide in therecombinant host cell.

Isolation and purification of polypeptides produced in the systemsdescribed above can be carried out using conventional methods,appropriate for the particular system. For example, preparativechromatography and immunological separations employing antibodies, suchas monoclonal or polyclonal antibodies, can be used.

Polynucleotides can be synthesized in the laboratory, for example, usingan automatic synthesizer. An amplification method such as PCR can beused to amplify polynucleotides from either genomic DNA or cDNA encodingthe polypeptides.

Polynucleotides and fragments thereof of the invention can be used, forexample, as probes or primers to detect the presence of canine CD20polynucleotides in a, such as a biological sample. A biological samplecan be, e.g., lymph node or tissue aspirate, serum, whole blood, cellarsuspension, or fluid effusion. The ability of such probes tospecifically hybridize to polynucleotide sequences will enable them tobe of use in detecting the presence of complementary sequences in agiven sample. Polynucleotide probes of the invention can hybridize tocomplementary sequences in a sample such as a biological sample, forexample, lymph tissue, thereby detecting the presence or absence ofcanine CD20 polynucleotides in samples. Polynucleotides from the samplecan be, for example, subjected to gel electrophoresis or other sizeseparation techniques or can be dot blotted without size separation. Thepolynucleotide probes are preferably labeled. Suitable labels, andmethods for labeling probes are known in the art, and include, forexample, radioactive labels incorporated by nick translation or bykinase, biotin, fluorescent probes, and chemiluminescent probes. Thepolynucleotides from the sample are then treated with the probe underhybridization conditions of suitable stringencies.

Depending on the application, varying conditions of hybridization can beused to achieve varying degrees of selectivity of the probe towards thetarget sequence. For applications requiring high selectivity, relativelystringent conditions can be used, such as low salt and/or hightemperature conditions, such as provided by a salt concentration of fromabout 0.02 M to about 0.15 M at temperatures of from about 50° C. toabout 70° C. For applications requiring less selectivity, less stringenthybridization conditions can be used. For example, salt conditions fromabout 0.14 M to about 0.9 M salt, at temperatures ranging from about 20°C. to about 55° C. The presence of a hybridized complex comprising theprobe and a complementary polynucleotide from the sample indicates thepresence of the microbe or polynucleotide sequence in the sample.

Antibodies and Antibody Fragments

Antibodies, such as monoclonal and polyclonal antibodies, thatspecifically bind polypeptides of the invention are part of theinvention. These antibodies can be made by using a polypeptide or apolypeptide fragment that contains an epitope present in a polypeptideshown in SEQ ID NO:6, SEQ ID NO:10 or SEQ ID NO:11 as an immunogen instandard antibody production methods (see e.g., Kohler et al., Nature,256:495, 1975; Ausubel et al. (1992) Current Protocols in MolecularBiology, John Wylie and Sons, Inc. New York, N.Y.; Harlow and Lane, Eds,(1988) Current Edition, Antibodies: A Laboratory Manual, Cold SpringHarbor Press, N.Y.). Antibodies can also be made using DNA immunizationtechniques using nucleic acid sequence coding for polypeptides SEQ IDNO:6, SEQ ID NO:10, SEQ ID NO:11 or fragments thereof in, e.g., standardmammalian expression vectors. See e.g., Chambers et al., 2003 NatureBiotechnology 21: 1088-1092; Tang et al. Nature. 1992 Mar. 12;356(6365):152-4; Barry et al., Biotechniques. 1994 April; 16(4):616-8,620.

An antibody is an intact immunoglobulin molecule, a fragment of animmunoglobulin molecule, such as Fab, Fab′, F(ab′)₂, F(ab)₂, Fv, sFv, ora single-chain antibody or fragments thereof, that specifically binds toa polypeptide of the invention (e.g., SEQ ID NO:6 or SEQ ID NO:10 andfragments thereof). Antibody fragments retain some ability toselectively bind to the antigen (e.g., a polypeptide of the invention)from which they are derived, and can be made using well known methods inthe art. In one embodiment of the invention, an antibody, antibodyfragment or single-chain antibody comprises all such antibodies thatspecifically bind to a polypeptide of the invention (e.g., SEQ ID NO:6or SEQ ID NO:10 and fragments thereof).

An antibody of the invention can be any antibody class, including forexample, IgG, IgM, IgA, IgD and IgE. An antibody or fragment thereofbinds to an epitope of a polypeptide of the invention. An antibody canbe made in vivo in suitable laboratory animals or in vitro usingrecombinant DNA techniques. Means for preparing and characterizingantibodies are well know in the art. See, e.g., Dean, Methods Mol. Biol.80:23-37 (1998); Dean, Methods Mol. Biol. 32:361-79 (1994); Baileg,Methods Mol. Biol. 32:381-88 (1994); Gullick, Methods Mol. Biol.32:389-99 (1994); Drenckchahn et al. Methods Cell, Biol. 37:7-56 (1993);Morrison, Ann. Rev. Immunol. 10:239-65 (1992); Wright et al. Crit. Rev.Immunol. 12:125-68 (1992). For example, polyclonal antibodies can beproduced by administering a polypeptide of the invention to an animal,such as a human or other primate, mouse, rat, rabbit, guinea pig, goat,pig, dog, cow, sheep, donkey, or horse. Serum from the immunized animalis collected and the antibodies are purified from the plasma by, forexample, precipitation with ammonium sulfate, followed bychromatography, such as affinity chromatography. Techniques forproducing and processing polyclonal antibodies are known in the art.

“Specifically binds” or “specific for” means that the polypeptiderecognizes and binds to an antibody of the invention with greateraffinity than to other, non-specific molecules. For example, an antibodyraised against an antigen (e.g., a polypeptide) to which it binds moreefficiently than to a non-specific protein can be described asspecifically binding to the antigen. Binding specifically can be testedusing, for example, an enzyme-linked immunosorbant assay (ELISA), aradioimmunoassay (RIA), or a western blot assay using methodology wellknown in the art.

Antibodies of the invention can be present in an antibody fusionprotein. An antibody fusion protein refers to a recombinant moleculethat comprises an antibody component and a therapeutic agent. Examplesof therapeutic agents suitable for such fusion proteins includeimmunomodulators (“antibody-immunomodulator fusion protein”) and toxins(“antibody-toxin fusion protein”).

Polypeptides of the invention comprise at least one epitope. An epitopeis an antigenic determinant of a polypeptide. Epitopes within apolypeptide of the invention can be identified by several methods. See,e.g., U.S. Pat. No. 4,554,101; Jameson & Wolf, CABIOS 4:181-186 (1988).For example, a polypeptide of the invention can be isolated andscreened. A series of short peptides, which together span the entirepolypeptide sequence, can be prepared by proteolytic cleavage. Bystarting with, for example, 100-mer polypeptide fragments, each fragmentcan be tested for the presence of epitopes recognized in, for example,an enzyme-linked immunosorbent assay (ELISA). In an ELISA assay apolypeptide, such as a 100-mer polypeptide fragment, is attached to asolid support, such as the wells of a plastic multi-well plate. Apopulation of antibodies are labeled, added to the solid support andallowed to bind to the unlabeled antigen, under conditions wherenon-specific adsorption is blocked, and any unbound antibody and otherproteins are washed away. Antibody binding is detected by, for example,a reaction that converts a colorless indicator reagent into a coloredreaction product. Progressively smaller and overlapping fragments canthen be tested from an identified 100-mer to map the epitope ofinterest.

Antigens that can be used in producing antibodies of the inventioninclude polypeptides and polypeptide fragments of the invention. Apolypeptide used to immunize an animal can be obtained by standardrecombinant, chemical synthetic, or purification methods. As is wellknown in the art, in order to increase immunogenicity, an antigen can beconjugated to a carrier protein. Commonly used carriers include keyholelimpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), andtetanus toxoid. The coupled peptide is then used to immunize an animal(e.g., a mouse, a rat, or a rabbit). In addition to such carriers, wellknown adjuvants can be administered with the antigen to facilitateinduction of a strong immune response.

Polyclonal and monoclonal antibodies can be purified, for example, bybinding to, and elution from, a matrix containing a polypeptide orpolypeptide fragment of the invention to which the antibodies wereraised. Additional methods for antibody purification and concentrationare well known in the art and can be practiced with the antibodies ofthe invention. Anti-idiotype antibodies corresponding to polypeptides ofthe invention are also included in the invention, and can be producedusing standard methods.

An antibody and antigen (e.g., a polypeptide or polypeptide fragment ofthe invention) specifically bind to each other if they bind to eachother with greater affinity than to other, non-specific molecules. Forexample, an antibody raised against an antigen to which it binds moreefficiently than to a non-specific protein can be described asspecifically binding to the antigen.

In one embodiment of the invention an antibody of the inventionspecifically binds canine CD20. Antibodies of the invention can be used,for example, to detect canine CD20 polypeptides in a biological sample.Antibodies of the invention can be used in vitro or in vivo forimmunodiagnosis. The antibodies are suited for use in, for example,immunoassays in which they are in liquid phase or bound to a solid phasecarrier (e.g., a glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylase, natural and modified cellulose, polyacrylamide,agarose, or magnetite carrier). The antibodies used in such immunoassayscan be detectably labeled (e.g., with an enzyme, a radioisotope, afluorescent compound, a colloidal metal, a chemiluminescent compound, aphosphorescent compound, or a bioluminescent compound) using any ofseveral standard methods that are well known in the art. Examples ofimmunoassays in which the antibodies of the invention can be usedinclude, e.g., competitive and non-competitive immunoassays, which arecarried out using either direct or indirect formats. Examples of suchimmunoassays include radioimmunoassays (RIA), flow cytometry, andsandwich assays (e.g., enzyme-linked immunosorbent assays (ELISAs)).RT-PCR assays can also be used to quantitatively detect canine CD20.Detection of antigens using the antibodies of the invention can be doneusing immunoassays that are run in either forward, reverse, orsimultaneous modes, including immunohistochemical assays onphysiological samples. Other immunoassay formats are well known in theart, and can be used in the invention.

Antibodies of the invention can be chimeric antibodies, for example,humanized or caninized antibodies. A humanized antibody, like amouse-human chimeric antibody, can be prepared, for example, as follows:(1) isolate the gene encoding the antibody of the present invention fromantibody-producing mouse cells; (2) replace the constant region of the Hchain of the antibody with that of the human IgE; and (3) introduceinto, for example, mouse myeloma J558L cells (See, Neuberger et al.,Nature 314:268-270 (1985)). Alternatively, human antibodies or canineantibodies, for example, can be prepared by immunizing mice whose immunesystems have been replaced with that of humans or canines with apolypeptide or polypeptide fragment of the present invention.

Antibodies that specifically bind canine CD20 antigens (e.g., CD20polypeptides), are particularly useful for detecting the presence ofCD20 antigens in a sample, such as a lymph node or tissue aspirate,serum, whole blood, cellular suspension, or fluid effusion sample from acanine or feline. An immunoassay for CD20 antigen can utilize oneantibody or several antibodies. Immunoassay protocols can be based upon,for example, competition, direct reaction, or sandwich type assaysusing, for example, labeled antibody. Antibodies of the invention can belabeled with any type of label known in the art, including, for example,fluorescent, chemiluminescent, radioactive, enzyme, colloidal metal,radioisotope and bioluminescent labels.

Antibodies of the invention or fragments thereof can be bound to asupport and used to detect the presence of a CD20 antigen. Supportsinclude, for example, glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses and magletite.

Antibodies of the invention can also be used in immunolocalizationstudies to analyze the presence and distribution of a polypeptide of theinvention during various cellular events or physiological conditions.Antibodies can also be used to identify molecules involved in passiveimmunization and to identify molecules involved in the biosynthesis ofnon-protein antigens. Identification of such molecules can be useful invaccine development. Antibodies of the invention, including, forexample, monoclonal antibodies and single chain antibodies, can be usedto monitor the course of amelioration of a disease. By measuring theincrease or decrease of CD20-positive cells in a test sample from ananimal, it can be determined whether a particular therapeutic regimentaimed at ameliorating the disorder is effective.

Methods of Treatment

Antibodies of the invention can used to treat canine CD20+ B-celllymphoma, immune-mediated hemolytic anemia, immune-mediatedthrombocytopenia, and systemic lupus erythematosus (SLE).

The invention also encompasses multimodal therapeutic methods whereinanti-CD20 antibody administration is supplemented with chemotherapy, orby administration of therapeutic proteins, such as immunoconjugates andantibody fusion proteins.

In general, the dosage of administered anti-CD20 antibodies, anti-CD20antibody components, immunoconjugates, and fusion proteins will varydepending upon such factors as the canine's age, weight, sex, generalmedical condition and previous medical history. Typically, it isdesirable to provide the recipient with a dosage of antibody component,immunoconjugate or fusion protein which is in the range of from about 1pg/kg to 10 mg/kg (amount of agent/body weight of canine), although alower or higher dosage also may be administered as circumstancesdictate.

Administration of antibody components, immunoconjugates or fusionproteins to a patient can be intravenous, intraarterial,intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal,by perfusion through a regional catheter, or by direct intralesionalinjection. When administering therapeutic proteins by injection, theadministration can be by continuous infusion or by single or multipleboluses.

Compositions of the invention can comprise anti-CD20 antibodies and apharmaceutically acceptable buffer, for example, sterile saline, sterilebuffered water, propylene glycol. Methods for preparing administrableagents, such as parenterally administrable agents, are described inPharmaceutical Carriers & Formulations, Martin, Remington'sPharmaceutical Sciences, 15th Ed. (Mack Pub. Co., Easton, Pa. 1975),which is incorporated herein by reference.

All patents, patent applications, and other scientific or technicalwritings referred to anywhere herein are incorporated by reference intheir entirety. The invention illustratively described herein suitablycan be practiced in the absence of any element or elements, limitationor limitations that are not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising”,“consisting essentially of”, and “consisting of” may be replaced witheither of the other two terms. The terms and expressions which have beenemployed are used as terms of description and not of limitation, andthere is no intention that in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by embodiments, optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

EXAMPLES Example 1

Cloning Canine CD20:

The nucleotide sequence for canine CD20 has not been published ordeposited in NCBI/GenBank. Using primers deposited in GenBank from anunpublished study using sequence tagged sites in the canine genome(Accession: L77424, L77425), a portion of the gene sequence for canineCD20 was amplified from canine peripheral blood mononuclear cell (PBMC)cDNA (SEQ ID NO:1 FIG. 1). Specifically, PBMCs were purified from 8 mlof canine whole blood using Ficoll-Paque PLUS according to themanufacturer's instructions (Amersham Biosciences). The PBMCs wereresuspended in Trizol (Invitrogen) and RNA was harvested as described inthe product insert. Using 2.5 μg of this RNA and Thermoscript RT(Invitrogen), canine PBMC cDNA was generated using oligo-dT primersaccording to the product insert. From this gene sequence, primers weredesigned for use with 5′ and 3′ RACE (Rapid Amplification of cDNA Ends)using a commercially available kit (Smart RACE, BDBiosciences—Clontech). The 5′RACE was successful using the kit accordingto the manufacturer's instructions. However, the 3′ RACE product wasincomplete, likely due to secondary structure in the mRNA. The completeelucidation of the 3′ sequence required the use of a more temperaturestable reverse transcriptase (Thermoscript RT, Invitrogen). The 3′ RACEcDNA was synthesized from 2.5 μg of canine RNA with 11 of ThermoscriptRT at 60° C. for 15 minutes, followed by incubation at 65° C. for 30minutes. This modification to the existing RACE technique resulted in acomplete 3′ sequence for canine CD20. The full length nucleotide andtranslated polypeptide sequences are shown in FIGS. 3 and 4 (SEQ ID NO:5 and 6), respectively. An alignment of the full-length sequences forhuman, mouse, feline and canine CD20 is shown in FIG. 5. Canine CD20 isonly 74% identical to human CD20 and 68% identical to murine CD20.

Example 2

Expression of Canine CD20:

The full-length nucleic acid sequence for canine CD20 was amplified fromcanine cDNA using a proof-reading Taq (High Fidelity Taq, Roche) andsubcloned into a commercially available expression vector (pCMV4AFLAG;Stratagene) at the NotI/EcoRI sites using standard molecular biologyprocedures. The expression plasmid was transformed into E. coli andpurified using a Maxi-Prep kit from Qiagen. The purified plasmid wastransiently transfected into COS7 cells (ATCC CRL-1651) using eitherLipofectamine (Invitrogen) or FuGene6 (Roche) according to themanufacturer's instructions. Two days post-transfection, the cells werewashed twice with phosphate buffered saline (PBS, pH 7.2), scraped fromthe surface of the well in buffer, and pelleted by centrifugation. Theresulting cell pellet was lysed in a Triton X-100 lysis buffer (50 mMTris (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, 2 mM EDTA, ProteaseInhibitor V (CalBiochem)) for 30 minutes on ice followed bycentrifugation of cell debris at 4° C. for 15 minutes. For total celllysate, the resulting supernatant was boiled in an equal volume of SDSsample buffer and loaded onto 4-20% SDS-PAGE gels (Pierce). Forimmunoprecipitation, the lysate was incubated with either 5 ul ofanti-FLAG M2 monoclonal antibody, 2.5 ul of monoclonal anti-CD20antibody (Biogenex MU265-UC and MU238-UC), or 5 ul of polyclonalanti-CD20 (Santa Cruz Biotechnology sc-15361, sc-7736, or LabVisionRB-9013) overnight at 4° C. with mixing. A 1:1 slurry of Protein Gsepharose (Amersham) in lysis buffer was added to the lysate for 30minutes at 4° C. with mixing. The antibody-bound sepharose was collectedby microcentrifugation and washed with 1 ml of lysis buffer three times.The washed sepharose was boiled in 1× sample buffer before being loadedonto a 4-20% SDS-PAGE gel (Pierce). The gel was then transferredovernight to PVDF (Millipore) for blotting.

Expression was confirmed on Western blots using an anti-FLAG M2monoclonal antibody (Stratagene) at 1:3000 in TBS/1% Casein (Pierce) andchemiluminescent detection (ECL, Amersham) (FIG. 6). A single band ofapproximately 40 kDa was observed for the total cell lysate preparation.Only the anti-FLAG monoclonal antibody and the polyclonal antisera fromLabVision were successful in immunoprecipitating canine CD20 from thetotal cell lysate (FIG. 6).

Cellular expression of canine CD20 was also demonstrated usingimmunofluorescence with the anti-FLAG antibody. COS7 cells transientlytransfected with canine CD20, as described above, were grown in two-wellchamber slides (Nunc). At 48 hours post-transfection, the cells werewashed twice with PBS, and fixed in 4% neutral buffered formalin for 15minutes. Slides were washed three times in PBS for 5 minutes each,followed by a 15 minute incubation with 50 in M ammonium chloride inPBS. After three five minute washes in PBS, the slides were blocked for1 hour with PBA (PBS, 0.1% Triton X-100, 15% normal goat serum, 1% BSA).Anti-FLAG M2 antibody (Stratagene) was diluted 1:200 in PBA and added tothe slides upon removal of the blocking solution and incubated for 1hour at room temperature. Slides were washed three times for 5 minutesin PBS before adding a goat anti-mouse FITC conjugated secondaryantibody (Jackson ImmunoResearch) at a 1:250 dilution in PBA. Thesamples were incubated for 1 hour at room temperature and again washedthree times for five minutes in PBS. Confocal microscopy was performedto evaluate surface expression of canine CD20. FIG. 7 shows the resultsof the immunofluorescent labeling and surface expression of canine CD20.

Example 3

Use of Canine CD20 for Peptides:

Based on the amino acid sequence of the canine CD20, and alignment withCD20 proteins from other species, a polypeptide sequence was identifiedthat represents the predominant extracellular domain of the canine CD20protein. This 53-mer polypeptide was synthesized alone (SEQ ID NO:10;FIG. 8) and in conjunction with a murine T-cell epitope from ovalbumin(SEQ ID NO:11; FIG. 8). The latter polypeptide was conjugated to KLH andBSA and was used to immunize mice in order to generate monoclonalantibodies to the extracellular domain of canine CD20. The formerpolypeptide was used in an ELISA to screen hybridomas for a monoclonalantibody specific to the extracellular domain of canine CD20.

The peptide ELISA was performed by dissolving 1 mg of the 53-mer peptide(SEQ ID NO:10) in 1 ml of DMSO (Sigma). Peptide was coated onto 96-wellmicrotiter plates (Immunlon 4HB, Dynatech) at a concentration of 10ug/ml in 50 mM carbonate (pH 9) overnight at room temperature. Plateswere washed four times in PBS-T (phosphate buffered saline (pH 7.2),0.05% Tween-20) and blocked with 2% Tween-20 in 100 mM Tris (pH7.4) for2 hours at room temperature. Plates were washed four times in PBS-Tbefore samples were added either neat, for hybridoma supernatants, ordiluted in sample diluent (50 mM Tris (pH 7.2), 0.05% Tween-20, 50%fetal bovine serum), for serum samples. Plates were incubated for 1 hourat room temperature, washed four times in PBS-T, and a 1:2500 dilutionof goat anti-mouse HRPO (Jackson ImmunoResearch) in sample diluent wasadded. Following a 1 hour incubation at room temperature, plates werewashed six times in PBS-T and developed with at TMB substrate (Moss,Inc.).

Example 4

Production of Monoclonal Antibodies to Canine CD20

Purified vector DNA (MaxiPrep Kit, Qiagen) containing the canine CD20gene (Example 2) was used for DNA immunization of mice according topublished protocols (Ulmer, J. B. et al. Science, 1993). Antibody titersfrom individual mice were evaluated ten days after the secondimmunization using the peptide ELISA described in Example 3. Positivetiters were found for each DNA-immunized mouse (FIG. 9). A thirdinjection was performed three weeks after the second injection and thespleen was harvested within 7-10 days for the fusion. The spleen wasfused with a mouse myeloma cell line FO using methods well know to thoseskilled in the art (see Antibodies, a Laboratory Manual, by Harlow andLane, Cold Spring Harbor Laboratory Press, 1988, pp 139-238). Individualmonoclonal antibody producing clones were isolated using the process oflimited dilution and screened on the peptide ELISA described in Example3. A total of 26 clones were isolated from the screening, all of whichproduced IgM antibodies.

Example 5

Immunocytochemical Evaluation of Monoclonal Antibodies to Canine CD20

The 26 identified clones reactive to the extracellular domain of canineCD20 were further evaluated on smears of canine lymph node aspiratesusing immunocytochemical techniques. Each smear was outlined with anImmEdge pen (Vector Labs), allowed to dry, and then fixed in acetone for3 minutes. Following a 5 minute wash in PBS (pH 7.2), slides weretreated with ammonium chloride (50 mM in PBS) for 15 minutes at roomtemperature. Following a 5 minute wash in PBS, slides were blocked inPBS/NGS (PBS with 15% normal goat serum (Vector Labs)) for 30 minutes atroom temperature. Each hybridoma supernatant was then added to the slideand incubated for one hour at room temperature. Slides were washed twicefor 5 minutes in PBS, followed by a 30 minute incubation in a 1:200dilution of goat anti-mouse IgM+IgG (H+ L) F(ab)₂ FITC (JacksonImmunoResearch) in PBS/NGS. Following two 5 minute washes in PBS, slideswere examined using fluorescent microscopy. Of the 26 clones initiallyidentified, only five clearly demonstrated immunofluoresence on thelymph node aspirates.

Example 6

Flow Cytometric Evaluation of Monoclonal Antibodies to Canine CD20 inCanine Lymphoma

Of the five clones identified, four were propagated in 1 L bioreactorbags (VectraCell) according to the manufacturer's instructions usinghybridoma serum-free media (Invitrogen). IgM antibodies were purifiedfrom the culture supernatants using a HiTrap IgM column (AmershamBiosciences) according to the product insert (FIG. 10). Purifiedantibodies were conjugated to NHS-Fluoroscein (Sigma/Fluka) by mixing a20 molar excess of conjugate with the antibody and allowing this toincubate for 30 minutes at room temperature. Antibodies were thenpurified from unreacted NHS-Fluoroscein using a microspin desaltingcolumn (Zeba Spin Column, Pierce) and PBS (pH 7.2). Of the fourantibodies that were fluorescently tagged, only two showed significantlabeling of lymphocytes by flow cytometry on lymph node aspirates fromdogs with lymphoma. The cell lines secreting these antibodies have beendeposited with the ATCC, Manassas Va. on Mar. 30, 2005. Straindesignations are F3C7 and F7A5, bearing ATCC Patent Deposit NumbersPTA-6661 and PTA 6662, respectively. For flow cytometry, lymph nodeaspirates were collected into 1 ml of media (Hanks Balanced SaltSolution (HBSS), 30 mM HEPES, 2% fetal bovine serum, K₃EDTA, Pen/Strep)and stored at 4° C. An aliquot of 100 ul was blocked with 25 μg of mousegamma globulin (Jackson ImmunoResearch) for 20 minutes on ice. The cellswere then incubated for 30 minutes on ice with approximately 60 μg ofeither F3C7 or F7A5. Controls included unlabeled cells, cells labeledwith a fluoroscein isotype control, cells labeled for a standard B-cellmarker (5 μl anti-CD79a-RPE, DAKOCyotmation) and an RPE isotype control(5 μl IgG1-RPE, DAKOCyotmation). Cells were washed twice with BD StainBuffer (BD Biosciences) and fixed in BD Cytofix for 15 minutes on ice,followed by two washes in BD Stain Buffer. Labeled cells were analyzedon a Becton Dickinson Flow Cytometer. Representative examples of theanti-canine CD20 monoclonal antibodies labeling B-cell lymphomas areshown in FIGS. 11 and 12.

Example 7

Use of Monoclonal Antibodies to the Extracellular Domain of Canine CD20in a Point of Care Hematology Instrument to Identify B-Lymphocytes.

Monoclonal antibody F7A5 was labeled with 60 nm colloidal gold(BBInternational). The pH of the gold was initially adjusted to pH 9with 100 mM K₂CO₃. The F7A5 monoclonal antibody was diluted toapproximately 2 mg/ml in 2 mM borate (pH 9) and was added dropwise tothe gold with stirring to a final concentration of 12 μg/ml. After 15minutes the gold-antibody solution was stabilized with a 1:10 dilutionof 10% BSA (pH 9). After 15 minutes of mixing the gold was centrifugedand washed three times (1% BSA, 1 mM NaCl, pH 9) and resuspended in thewash buffer to a final OD of 5. A lymph node aspirate from the poplitealnode of a healthy, young dog was obtained and resuspended in thecollection media described in Example 6. A 100 μl aliquot of this samplewas incubated with 10 μl of the colloidal gold labeled F7A5 monoclonalantibody to canine CD20 for 30 minutes at room temperature. The samplewas then diluted in 0.5 mls of PBS immediately prior to analysis. As acontrol, an unlabeled aliquot of identical volume from the same aspiratewas analyzed in 0.5 ml of PBS.

Samples were analyzed on a point of care hematology instrument(LaserCyte, IDEXX Laboratories, Inc.) under routine conditions,substituting the standard sheath solution for PBS. See, e.g., U.S. Pat.Publ. No. 2004/0246480. Data obtained from the samples was analyzedusing standard flow cytometry software (FCS Express 2, De NovoSoftware). Initially, small lymphocytes are gated in the forward scatterhigh (FSH) vs time of flight channel (TOF). Medium lymphocytes can beidentified in the extinction integral (EXTint) channel vs. TOF. Afterselecting for each of these populations, the data can be analyzed fordegree of right angle scatter, comparing the unlabeled control to theF7A5 labeled sample. Due to the light scattering properties of colloidalgold, cells labeled with the gold-tagged F7A5 antibody demonstrateincreased scatter in the right angle scatter channel (RAS). FIG. 13shows that approximately 33% of small to medium lymphocytes areidentified as B-lymphocytes in this normal lymph node aspirate from adog. This value is consistent with the values obtained usingfluorescently labeled antibodies to canine B-cells on standard flowcytometry as reported in the literature (D. Gibson et al., JVIM (2004)18:710-17).

Example 8

Use of F7A5 Monoclonal Antibody to Identify B-Lymphocytes in a LymphNode Aspirate from a Cat with Lymphoma

A lymph node aspirate from a cat with lymphoma was collected andresuspended in collection media as described in Example 7. A 100 μlaliquot of the sample was labeled with 10 μl of the colloidal goldlabeled F7A5 monoclonal antibody for 60 minutes at room temperature.Following labeling the sample was diluted in 1 ml of PBS for analysis asdescribed in Example 7. The lymphocyte populations were determined in amanner similar to that described for Example 7 and the gated lymphocytepopulation is shown in FIG. 14. Compared to the unlabeled controlsample, at least 12.5% of the gated lymphocytes are CD20 positiveB-cells.

1. An isolated antibody or antigen binding portion thereof thatspecifically binds SEQ ID NO:6 or SEQ ID NO:10.
 2. The isolated antibodyor antigen binding portion thereof of claim 1, wherein the isolatedantibody or antigen binding portion thereof is a monoclonal antibody, apolyclonal antibody, or single chain antibody.
 3. The isolated antibodyof claim 2, wherein the antibody is produced by myeloma cell line ATCCPTA-6661 or ATCC PTA-6662.
 4. The antigen binding portion thereof ofclaim 2, wherein the antigen binding portion thereof is a Fab fragment,a F(ab′)₂ fragment, a Fab′ fragment, a F(ab)₂ fragment, a sFv fragment,a single chain antibody, or a Fv fragment.
 5. The isolated antibody orantigen binding portion thereof of claim 1, wherein the antibody orantigen binding portion thereof belongs to an antibody class selectedfrom the group consisting of IgG, IgM, IgA, IgD and IgE.
 6. An isolatedantibody or antigen binding fragment thereof that (a) competes with areference antibody for binding to SEQ ID NOs:6 or 10 or antigen bindingfragments thereof; (b) binds to the same epitope of SEQ ID NOs:6 or 10or antigen binding fragments thereof as a reference antibody; (c) bindsto SEQ ID NOs:6 or 10 or antigen binding fragments thereof withsubstantially the same K_(d) as a reference antibody; or (d) binds toSEQ ID NOs:6 or 10 or antigen binding fragments thereof withsubstantially the same off rate as a reference antibody, wherein thereference antibody is an antibody or antigen binding fragment thereofthat specifically binds to a polypeptide of SEQ ID NOs:6 or 10 orantigen binding fragments thereof with a binding affinity K_(a) of 10⁷l/mol or more.
 7. An isolated antibody or antigen binding fragmentthereof that competes with the isolated antibody of claim 3 for bindingto SEQ ID NOs:6 or 10 or antigen binding fragments thereof.